Hyaluronan (HA, hyaluronate, hyaluronan, hyaluronic acid) is a negatively charged, high molecular weight, connective tissue polysaccharide found in the extracellular matrix of most animal tissues. HA consists of alternating N-acetyl-D-glucosamine and D-glururonic acid residues linked by B(1–4) and B (1–3) bonds which has a molecular weight ranging from 1 and 50×106 Da (Brimacombe, JS., et al., in Mucopolysaccarides. (Elsevier, Amsterdam, 1964)) depending upon its source. For example, its has been determined that HA averages between 3–5×106 Da, or 6–7×106 Da, when isolated from rheumatoid fluid, or normal synovial fluid, respectively (Laurent, TC, et al., Immunol Cell Biol., 74:1–7, (1996)). In addition, dilute solutions of HA (<1 mg/mL) are known to result in highly entangled networks which instill unique rheological characteristics to the solution in hand (Laurent, T C., Immuno Cell Biol., 74:1–7, (1996)). For example, solutions of hyaluronan are viscoelastic with the viscosity maintaining a pronounced dependency on shear forces (Ogston, A G., et al., J. Physiol., 199:244–52, (1953)). Therefore, considering the increased localization of HA in the body between surfaces that move against each other, combined with the mechanical/physical characteristics ascribed above, HA has been attributed the primary role of lubrication and protection of joints and tissues, cartilage surfaces and muscle bundles. Further, HA has also been associated with the scavenging of free radicals and debris (Myint, P., et al., Biochim. Biophys. Acta, 925:194–202, (1987), and Laurent, T C, Ann. Rheum. Dis., 54:429–32, (1995), respectively), keeping the joint cavities open (Edwards, J C W., et al., J. Anat., 185:355–67, 1994), forming flow barriers in the synovium (McDonald, J N., et al., J. Physiol., 485.1:179–93, (1995)), and the prevention of capillary growth (Sattar, A., Semin. Arthritis Rheum., 22:37–43, (1992)).
HA is synthesized ubiquitously in the plasma membrane of all vertebrate tissues and in some bacteria (Fraser, J R E, J. Intern Med., 242:27–33, (1997)). It is catabolized locally through receptor-mediated endocytosis and lysosomal degradation, in addition to, the lymph nodes and endothelial cells of the liver sinusoids. HA is commonly isolated from the vitreous body of the eye, synovial fluid, rheumatoid fluid, umbilical cord, and skin. Several physiological functions have been associated with HA, in particular water homeostasis; mitosis, cell migration, differentiation, angiogenesis (Rooney P and Kumar S (1994) EXS (Switzerland) 70:179–90); and tissue remodeling, both in normal or tumor-associated events. Its role in water homeostasis (resistance to bulk flow of solvent) is particularly important as it has been shown to prevent excessive fluid exchange between tissue compartments, during both normal conditions and injury (Day, T D., Nature, 166:785–6., (1950)). In addition, HA is thought to play an important role in the promotion of cell proliferation and migration during tissue development and regeneration (Toole, B P., in Cell Biology of Extracellular Matrix (Hay ED, ed), pp. 305–339 (Plenum Press, New York, (1991)).
The matrix-induced effects on cells are directed by a wide variety of HA-binding proteins which are classified into two groups: structural (matrix) and cell-surface-associated (HA-receptors) (Tool, B P., Curr Opin Cell Biol 2:839–844 (1990)). The widespread occurrence of HRs indicate their importance in tissue organization and control of cellular behavior. The family is known as the hyaladherins and includes those RA-binding proteins which act as part of the structural matrix and those which interact with HA at the plasma membrane as cell-surface matrix receptors. Although not comprehensive, some of the identified members of the hyaladherin family include aggrecan, link protein (Manuskiatti, W., Int J Dermatology, 35(8):539–533, (1996)), versican, hyaluronectin, neurocan (Knudson, C B et al., FASEB J, 7:1233–1241, (1993)), CD44 family of receptors (Underhill, C B., J Cell Sci,), RHAMM (Receptor for Hyaluronan-Mediated Motility), and TSG-6 (Tumor Necrosis Factor-Stimulated Gene 6). With the recognition of the Hyaluronan cell-surface receptor (HR), cell biologists, pathologists, and immunologists have begun to investigate the importance of the HA and HR for their potential diagnostic and therapeutic value.
HRs found within the cartilage matrix have been well characterized. Aggrecan is the large aggregating chondroitin sulfate proteoglycan of cartilage which has a high affinity for HA (Hardingham et al, Biochim Biophys. Acta., 279:401–405, (1972)). Link protein is a 45–48 kDa glycoprotein which also demonstrates strong specific binding affinity. HA may bind more than 100 aggrecan and link protein molecules in a supramolecular complex which confers the viscoelastic properties of cartilage. Other matrix proteins such as PG-M and type VI collagen which participate in assembly and integrity may also be involved.
HA-binding proteins are also found in noncartilaginous tissues. Versican of fibroblasts, hyaluronectin of nervous and soft connective tissues, glial hyaluronan binding protein in the central nervous system, and neurocan, a chondroitin sulfate proteoglycan of brain, also form strong structural complexes with HA. All matrix hyaloadherins contain tandem repeated B loops, a structural motif believed to contain the HA-binding domain.
HR hyaloadherins have been detected on several cell types from a wide variety of tissues based upon hyaluronans ability to aggregate such cells (Pessac, B., et al., Science, 175:898–900, (1972)). Some reports suggest that HRs are related to the CD44 family of lymphocyte homing receptors which include the isoforms, Pgp-1, Hermes antigen, H-CAM and ECMRIII. The distal extracellular domain of CD44 has sequence homology to one of the B loop motifs of link protein. The numerous isoforms suggest different cellular functions and demonstrate binding to other ligands such as collagens I and IV and mucosal vascular addressing. Further, although many roles have been attributed to the CD44-hyaluronan interaction, its roles in development, tumour progression, and in the immune response appear to be the most prevalent (Sherman, L., Curr. Opinion Cell Biol., 6:726–33, (1994).
Other non-CD44 HRs include cell-surface antigens termed IVd4 which block binding of HA, liver endothelial cell receptors (LEC) which are involved in the clearance of HA from the circulation, and fibroblast-produced HR which may be located on the cell surface where it mediates HA-induced cell locomotion. Its 58 kDA soluble form contains an HA-binding component unrelated to the B loop motif and is known as a receptor for HA mediated motility (RHAMM). The important distinctions between cell-surface and matrix hyaloadherins are 1) HA hexasaccharides represent the minimum size molecule that interacts with these cell-surface receptors, 2) binding affinity increases with increasing polymer length, and 3) binding increases with increasing buffer ionic strength.
Increased matrix presence of HA has been correlated with cell migration in embryogenesis, limb regeneration, wound healing and tumor invasion. Since the CD44 HR have been shown to associate with cytoskeletal ankyrin, proteins of the HR complex may affect re-organization of the actin cytoskeleton and other activities such as cell ruffling, detachment from the substratum, and locomotion necessary for cell migration. RHAMM, as one of the HR complex proteins, binds to HA with high affinity and is expressed only in the leading lamellae and perinuclear regions of migrating fibroblasts.
Since RHAMM does not include a transmembrane hydrophobic region, it is assumed to be a peripheral protein associated with intracellular, membrane bound tyrosine kinase. In studies of timed administration of HA and an inhibitor of tyrosine kinase, HA stimulated locomotion via a rapid tyrosine kinase signal transduction pathway.
Invasive or metastatic cancer cells have the capacity to exit from the vascular system by use of sets of molecules, at least one of which always has a receptor function. One series of such sets might include successive interactions among endothelial VLA-4 integrin and E-selectin, subendothelial collagen IV and B-4 integrin, and soft connective tissue HA and CD44 or HR interactions (Zetter BR (1993) Semin Cancer Biol 4:215–218).
Some tumor cells also have the capacity to assemble HA-enriched pericellular matrices which reduce cell adhesion to the outside of the growing tumor and protect the tumor from immune surveillance. In addition, the presence of high HA attracts endothelial cells which are active in angiogenesis. The combination of these HA functions allows the rapid establishment and growth of invasive tumor cells.
The transforming oncogene H-ras may promote cell locomotion. Hardwick et al (1992 J Cell Biol 117:1343–1350) reported that H-ras actually regulates expression of RHAMM, showed binding between HA and RHAMM, and produced an antibody to the protein which is capable of inhibiting HA/HR locomotion.
The fact that WF-HABP, OE-HABP, and BM-HABP polynucleotides and polypeptides are members of the hyaluronan receptor family suggests that: invention would play an important role in diverse human disease states ranging from inflammatory conditions to, cancer metastasis, and more generally that members of this family mediate cellular responses such as activation, survival, proliferation, migration, signalling, and differentiation; that hyaluronan receptor family members provide an important model system for the in vitro study of arthritus, angiogenesis, and hematopoietic or immune disorders; and that hyaluronan receptors would provide defined targets for the development of new anti-cancer, arthritus, and healing wound tissue agents.